ENZYMES

Internship Clinical Didactics

Gabriel G. Ponce de Leon,RMT,AMT,MLS(ASCPi)
Clinical Instructor
What are enzymes?
• Protein catalyst of biochemical reaction

• NO or minimal change

• High temperatures

• Acidic pH

• Physical destruction

• Requires a lot of energy

What are enzymes?
• Enzymes reduces the energy required for a
reaction to occur

• = reduced energy of activation

Rate of reaction
EOA

0 Reaction
Enzymes as biological catalyst

• E + S  ES or ES  E + P

• Example: Conversion of hydrogen peroxide

into oxygen and water

• Catalase: increases the reaction by

1,000,000,000
Active site

• Pocket of cleft formed by amino acids that

that participate in substrate binding and
catalysis

• Common amino acids: aspartate, glutamate

cysteine, serine, histidine and lysine
Enzyme kinetics

• To measure rate enzymatic reaction:

mix enzyme + substrate  record the
formation of the product or disappearance
of the substrate at neutral pH and @ temp
25 to 37 degrees celsius

Michaelis Menten equation

• V = Vmax(S)
--------
Km + S
Enzyme kinetics

• Vmax – used to measure the enzyme’s

catalytic efficiency
Enzymes
No reaction
40 Maximum
30 velocity

20

10

10 20 30 40 50 50 Substrates
Enzyme kinetics

• Km – Michaelis Menten constant – it

measures the affinity of an enzyme
Enzymes for a substrate

40
Substrate
concentration
30 at which
20 velocity is at
half maximal
10

10 20 30 40 50 50 Substrates
Enzyme inhibition

• What are enzyme inhibitors?

 Low molecular weight compounds

 Reduce or inhibit the enzyme catalytic

activity
 Can be either reversible and
irreversible
Enzyme inhibition

• What are enzyme inhibitors?

 Two types: specific and non-specific

 Has set of rules

RULES:
1.Enzyme interacts with substrate in 1:1
ratio at active site to catalyze reaction

2.Enzyme binds to substrate at active site

in the form of lock and key 3D arrangement
for induced fit
3.Inhibitor active groups compete with
substrate active groups and/or active
groups at enzyme allosteric catalytic
site in a synergistic manner or first
come first preference (competition) to
make EIS/ES/EIC
RULES:
4.Enzyme-Inhibitor-substrate complex
formation depends on active energy loss
and thermodynamic principles.

5.Enzyme and substrate or inhibitors react

with each other as active masses and
reaction progresses in kinetic manner
of forward or backward reaction.

6.Kinetic nature of inhibitor or substrate

binding with enzyme is expressed as
kinetic constants of a catalytic reaction
RULES:
7. Enzyme reaction(s) are highly depend on
physiological conditions such as pH,
temperature, concentration of reactants,
reaction period to determine the rate
of reaction

8. Substrate and inhibitor molecules arrange

over enzyme active site on specific
subunit(s) in 3D manner. As a result
enzyme-substrate-inhibitor exhibit
binding rates depend on allosteric sites
or subunit-subunit homotropic or
heterotropic interactions.
RULES:
9. Intermolecular forces between enzyme
subunits, substrate or inhibitor active
group interactions, physical properties
of binding nature: electrophilic,
hydrophilic, nucleophilic and
metalloprotein nature; hydrogen bonding
affect the overall enzyme reaction rates
and mode of inhibition (3D orientation of
inhibitor molecule on enzyme active site).
Enzyme inhibition

• Specific inhibitors – attack a specific

component of the holoenzyme system
Several forms:
 coenzyme inhibitors
 Inhibitions of ion cofactors
 Prosthetic group inhibitors
 Apoenzyme inhibitors
 Physiological modulators
Enzyme inhibition

• Competitive inhibition – inhibitor is

structurally similar with the substrate
COMPETES with the ACTIVE SITE
Alters the Km but not the Vmax
Example: sulfa drug
Resembles  p-aminobenzoic acid
NO folic acid production – death of
Bacterial cells
Enzyme inhibition
CLINICAL AND
LABORATORY
APPLICATION
OF ENZYMES
PANCREATIC ENZYMES

• Amylase
• Lipase
• Colipase
• Rnase
• DNase
PANCREATIC ENZYMES
AMYLASE
Normal value: 28-100 U/L
Causes of raised plasma amylase
Marked increase:
 Acute pancreatitis
 Severe glomerular impairment
 Perforated peptic ulcer
Pancreatic pseudocyst – plasma amylase
activity fails to fall after an attack of
acute pancreatitis
PANCREATIC ENZYMES
AMYLASE
Macroamylasaemia – high plasma amylase
Activity is due to low renal excretion of
enzyme
PANCREATIC ENZYMES
LIPASE
Presence of bile salts is required a
Cofactor (colipase)
Clinical Significance:
Normal values: 40 – 200 U/L
Marked increase:
 Acute pancreatitis
 Pancreatic carcinoma
LIVER ENZYMES
Three types of
MARKERS:
Marker of –
1. Hepatocellular damage
2. Cholestasis
3. Disturbance in synthesis
LIVER ENZYMES
Marker of Hepatocellular Damage
Aminotransferases
Requires cofactor: Pyridoxal PO4
AST (SGOT)
LIVER ENZYMES
Marker of Hepatocellular Damage
ALT (SGPT)
LIVER ENZYMES
Locations:
ALT (SGPT)
AST (SGOT)
LIVER ENZYMES
Increased:
ALT (SGPT) Male <45 U/L, Female <35 U/L
• Liver Disease (ex. Hepatitis)
• 100 – 1000 times increase in acute viral
hepatitis. (but ALT is higher than AST)
AST (SGOT) Male <35 U/L, Female <31 U/L
• Liver Disease
• Acute Myocardial Infarction
• Hemolysis
• Circulatory failure, shock and hypoxia
LIVER ENZYMES
Marker of Cholestasis

• Alkaline Phosphatase
• Gamma Glutamyl Transferase

• Glutamate Dehydrogenase
LIVER ENZYMES
Marker of Cholestasis
Alkaline Phosphatase (ALP)
• Requires a high pH
• High Concentration in osteoblasts

• Intestinal wall, hepatobiliary tract

increase: decrease:
• Physiological • Arrested bone growth
• Bone disease • hypophosphatasia
• malignancy
LIVER ENZYMES
Marker of Cholestasis
Alkaline Phosphatase (ALP)
ISOENZYMES:
• Liver, Bone, placenta, intestine
LIVER ENZYMES
Marker of Cholestasis
Gamma Glutamyl Transferase (GGT)
Male <55 U/L, Female <38 U/L
• Liver, kidney, pancreas and prostate
increase:
• Drugs or alcohol
• Viral hepatitis
LIVER ENZYMES
Marker of Cholestasis
Glutamate Dehydrogenase
Male <6 U/L, Female <8 U/L

• Liver, heart muscle, kidneys

• Released from necrotic cells
MUSCLE ENZYMES
• Creatine Kinase

• Lactate Dehydrogenase
(LD 1 and 2)
MUSCLE ENZYMES
Creatine Kinase
Male 46-171 U/L, Female <34-145 U/L
• Liver, heart muscle, kidneys
MUSCLE ENZYMES
Creatine Kinase
• Two subunits: Brain and Muscle
• Isoenzymes: CK-BB (CK1), CK-MB (CK2),
CK-MM (CK3)

increase:
• muscular dystrophy
• Viral myositis, polymyositis
MUSCLE ENZYMES
CK-MB (CK2)
• Accounts for 35% of
total CK in cardiac muscle
• Always high after MI

CK-BB (CK1)

• Brain, gastrointestinal,
genital tracts
CK-MM (CK3)
• Skeletal and cardiac muscle
MUSCLE ENZYMES
Lactate Dehydrogenase
Normal range: 180-360 U/L
• Interconversion of lactic to pyruvic
• cardiac and skeletal muscle, liver,
kidney, brain and erythrocytes

LD-1 (HHHH; H4) = migrates fastest

towards the anode
LD-2 (HHHM; H3M)
LD-3 (HHMM; H2M2)
LD-4 (HMMM; HM3)
LD-5 (MMMM; M4)
MUSCLE ENZYMES
Lactate Dehydrogenase
increase:
• Myocardial Infarction
• Acute leukemia
• Acute hepatitis
Increased LD1 and LD5: myocardial
infarction, in megaloblastic anaemia and
after renal infarction.
Increased LD2 and LD3: acute leukaemia
Increased LD5: damage to liver and skeletal
muscle
 OTHER CLINICALLY
ENZYMES IMPORTANT ENZYMES
Acid Phosphatase

Glucose 6 phosphate Dehydrogenase

 OTHER CLINICALLY
ENZYMES IMPORTANT ENZYMES
Acid Phosphatase
• Present in lysosomes

• Prostate, spleen, bone, erythrocytes,

platelets
• Lysosomal and prostatic ACP: TLACP

• Red Blood Cell and bone ACP: TRACP

 OTHER CLINICALLY
ENZYMES IMPORTANT ENZYMES
Increase (TRACP):
Normal range: 1.5-4.5 U/L
• Pagets disease

• Hyperparathyroidism

• Bone cancers
 OTHER CLINICALLY
ENZYMES IMPORTANT ENZYMES
Glucose 6 phosphate Dehydrogenase

• Expressed in all cells

• First step in the hexose monophosphate
shunt
• Generation of NADPH
The majority of G6PD –deficient individuals
develop hemolysis only when oxidative stress
occurs, as with infections and after
ingestion of certain drugs or fava beans.
FINISHED